Total RNA Extraction from the Aromatic Phalaenopsis bellina, Endemic Orchid in Sabah, Borneo
Keywords:Phalaenopsis bellina, Orchidaceae, RNA, Phenol-chloroform, Isolation, Flower
Phalaenopsis bellina is an attractive orchid due to its unique appearance and distinctive floral fragrance. Many past studies on this plant focused on the plant at the molecular level; however, this requires sufficient quantities of high-quality P. bellina RNA. RNA is more delicate to manipulate than DNA due to its structural instability and its vulnerability to various secondary metabolites, such as polyphenols and polysaccharides. Therefore, in this study, 4 RNA isolation methods, a modified phenol-chloroform method and 3 commercial kits (Vivantis, Novogene, and Analytik Jena) were used on the leaves and flowers of P. bellina for comparison. The yield and purity of the total RNA were determined using spectrophotometry. The results showed that the total RNA isolated using the modified phenol-chloroform method had the highest yield (1223.75±68.51 ng/µL) and purity compared to the 3 commercial kits, with an OD260/280 value of 2.07 and an OD260/230 value of 2.26, respectively. In particular, the isolated RNA did not show any detectable genomic DNA contamination or other impurities. The RNA isolated using the phenol-chloroform method was also evaluated by electrophoresis, reverse transcription, and PCR. The results indicated that the phenol-chloroform method appears to be superior for total RNA extraction. Thus, this developed method is proven to be suitable for the RNA extraction of plants rich in polysaccharides and polyphenols and is amenable for future molecular studies on P. bellina.
TB Beaman, JJ Wood, RS Beaman and JH Beaman. Orchids of Sarawak. Natural History Publications (Borneo) Sdn. Bhd., Kota Kinabalu, Sabah, Malaysia, 2001, p. 8-12.
AB Martin. The Vocabulary of Orchids: An Amateur Perpective. Australia, 2005, p. 116.
EA Christenson and MW Whitten. Phalaenopsis bellina (Rchb.f.) Christenson, A Segregate from P. violacea Witte (Orchidaceae: Aeridinae). Brittonia 1995; 47, 57-60.
M Mahmood and YF Chew. Agrobacterium-mediated genetic transformation of Phalaenopsis bellina using GFP and GUS reporter genes. Pertanika J. Sci. Technol. 2008; 16, 129-39.
YC Chuang, MC Lee, YL Chang, WH Chen and HH Chen. Diurnal regulation of the floral scent emission by light and circadian rhythm in the Phalaenopsis orchids. Bot. Stud. 2017; 58, 50.
YY Hsiao, WC Tsai, CS Kuoh, TH Huang, HC Wang, TS Wu, YL Leu, WH Chen and HH Chen. Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway. BMC Plant. Biol. 2006; 6, 14.
YY Hsiao, MF Jeng, WC Tsai, YC Chuang, CY Li, TS Wu, CS Kuoh, WH Chen and HH Chen. A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif. Plant J. 2008; 55, 719-33.
YC Chuang, YC Hung, CY Hsu, CM Yeh, N Mitsuda, M Ohme-Takagi, WC Tsai, WH Chen and HH Chen. A dual repeat cis-element determines the expression of geranyl diphosphate synthase for monoterpene production in Phalaenopsis orchids. Front Plant. Sci. 2018; 9, 765.
YC Chuang, YC Hung, WC Tsai, WH Chen and HH Chen. PbbHLH4 regulates floral monoterpene biosynthesis in Phalaenopsis orchids. J. Exp. Bot. 2018; 69, 4363-77.
L Liu, R Han, N Yu, W Zhang, L Xing, D Xie and D Peng. A method for extracting high-quality total RNA from plant rich in polysaccharides and polyphenols using Dendrobium huoshanse. PloS One 2018; 13, e0196592.
DV Jobes, DL Hurley and LB Thien. Plant DNA isolation: A methodology to efficiently remove polyphenolics, polysaccharides and RNA. Taxon 1995; 44, 379‐86.
Y Pico de Coaña, N Parody, E Fernández-Caldas and C Alonso. A modified protocol for RNA isolation from high polysaccharide containing Cupressus arizonica pollen: Applications for RT-PCR and phage display library construction. Mol. Biotechnol. 2010; 44, 127-32.
KK Sharma, M Lavanya and V Anjaiah. A method for isolation and purification of peanut genomic DNA suitable for analytical applications. Plant Mol. Biol. Rep. 2000; 18, 393,
D Yin, H Liu, X Zhang and D Cui. A protocol for extraction of high-quality RNA and DNA from peanut plant tissues. Mol. Biotechnol. 2011; 49, 187-91.
E Macrae. Extraction of Plant RNA. In: E Hilario and J Mackay (Eds.). Molecular Biology. Vol 353. Protocols for Nucleic Acid Analysis by Nonradioactive Probes. 2nd (eds). Humana Press, Totowa, New Jersey, USA, 2007, p. 15-26.
Kam-Lock Chan, CL Ho and P Namasivayam. A simple and rapid method for RNA isolation from plant tissues with high phenolic compounds and polysaccharides. Nat. Protoc. 2007; 2007, 184.
A Abdul-Rahman, NI Suleman, WA Zakaria, HH Goh, NM Noor and WM Aizat. RNA extractions of mangosteen (Garcinia mangostana L.) pericaps for sequencing. Sains Malays. 2017; 46, 1231-40.
A Das, D Saha and TP Mondal. An optimized method for extraction of RNA from teat roots for functional genomics analysis. Indian J. Biotechnol. 2013, 12, 129-32.
XB Ma and J Yang. An optimized preparation method to obtain high-quality RNA from dry sunflower seeds. Genet. Mol. Res. 2011; 10, 160-8.
MS Lewis, DJ Pikaard, M Nasrallah, JH Doelling and CS Pikaard. Locus specific ribosomal RNA gene silencing in nucleolar dominance. PLoS One 2007; 2, e815.
SN Palani, S Elangovan, A Menon, M Kumariah and J Tennyson. An efficient nucleic acids extraction protocol for Elettaria cardamomum. Biocatal. Agric. Biotechnol. 2019; 17, 207-12.
P Chomzynski and N Sacchi. Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987; 162, 156-9.
P Azizi, MY Rafili, M Mahmood, SNA Abdullah, MM Hanafi, MA Latif, M Sahebi and S Ashkani. Evaluation of RNA extraction methods in rice and their application in expression analysis of resistance genes against Magnaporthe oryzae. Biotechnol. Biotechnol. Equip. 2017; 31, 1-10.
L Meng and L Feldman. A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds. Biotechnol. J. 2010; 5, 183-6.
C Shu, S Sun, J Chen, J Chen and E Zhou. Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani. Electron. J. Biotechnol. 2014; 17, 50-4.
Ş Tüzmen, Y Baskin, A Feyda Nursal, S Eraslan, Y Esemen, G Çalibaşi, AB Demir, D Abbasoglu and C Hizel. Chapter 14 - Techniques for Nucleic Acid Engineering: The Foundation of Gene Manipulation. In: D Barh and V Azevedo (Eds.). Omics Technologies and Bio-engineering, Academic Press, India, 2018, p. 247-315.
JM Butler. Chapter 2 - DNA Extraction Methods. In: JM Butler (Eds.). Advanced Topics in Forensic DNA Typing: Methodology. Academic Press, San Diego, California, 2012, p. 29-47.
L Jaakola, AM Pirttilä, M Halonen and A Hohtola. Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit. Mol. Biotechnol. 2001; 19, 201-3.
DH Farkas and CA Holland. Chapter 3 - Overview of Molecular Diagnostic Techniques and Instrumentation. In: RR Tubbs and MH Stoler (Eds.). Cell and Tissue Based Molecular Pathology. Churchill Livingstone, Philadelphia, 2009, p. 19-32.
KM Elkins. Chapter 4 - DNA Extraction. In: KM Elkins (Eds.). Forensic DNA Biology. Academic Press, San Diego, California, 2013, p. 39-52.
SA Thatcher. 3 - Nucleic Acid Isolation. In: N Rifai, AR Horvath and CT Wittwer (Eds.). Principles and Applications of Molecular Diagnostics. Elsevier, USA, 2018, p. 35-46.
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